Hyperbranched polymeric micelles for encapsulation and delivery of hydrophobic molecules

ABSTRACT

Polymeric micelles for encapsulation of hydrophobic molecules are provided. Methods and formulations for delivering hydrophobic molecules to a host via these micelles are also provided. Methods of stabilizing liposomes or lipid based formulations by addition of polymeric micelles are also provided.

INTRODUCTION

This application is a continuation-in-part of U.S. application Ser. No.09/298,729, filed Apr. 23, 1999.

FIELD OF THE INVENTION

The present invention relates to new hyperbranched colloidal polymerswith micellar properties. The polymers comprise a mucic acid, alkylchains and poly(ethylene glycol). Hydrophobic molecules encapsulatedwithin these polymeric micelles are thermodynamically stable in aqueoussolutions, suspensions, dispersions, emollients, lotions, creams,salves, balms and ointments at ambient, refrigerated and elevatedtemperatures. Further, these polymers have been found to stabilizeliposomes and other lipid based structures used routinely in these typesof formulations for extended periods of time such that precipitation isprevented and optical transparency is maintained. While the polymers ofthe present invention can be used to encapsulate and deliver anyhydrophobic molecule, these colloids are particularly useful in deliveryof hydrophobic drugs via various routes and in the topical delivery ofhydrophobic molecules used routinely in cosmetics, toiletries,fragrances, perfumes, skin care products and beauty aids. Suchhydrophobic molecules include, but are not limited to, dyes, proteins,vitamins and fragrances.

BACKGROUND OF THE INVENTION

Micelles are a class of surfactants that form organized structures,referred to as colloidal spheres, in aqueous media. The hydrophobicshell of micelles makes the entire assembly water-soluble while thelipophilic core solubilizes hydrophobic molecules. Attempts have beenmade to use micelles in drug delivery applications because thelipophilic core serves as a microcontainer for drugs, therebysegregating the drugs from the outer environment by the hydrophilicsegments. Micelles or colloids encapsulate the drug, shielding the bodyfrom potentially toxic levels of drug while simultaneously protectingthe drug from inactivating agents in the blood and lymphatic system.Thus, solubilization of water-insoluble drugs by micelles has long beeninvestigated as a means for improving solubility for drug delivery, inparticular for parenteral or oral administration, and also forophthalmic, topical, rectal and nasal delivery (Florence, A. Techniquesof Solubilization of Drugs, Ed. Yalkowsky, S. (New York: Marcel Dekker,1981); Atwood, D. and Florence, A. T. Pharmaceutical Aspects ofSolubilization, Surfactant Systems. Their Chemistry, Pharmacy andBiology, (London: Chapman Hall, 1983):293-387).

However, the formation of micelles is both temperature- andconcentration-dependent. The concentration dependency is defined as thecritical micelle concentration or CMC. Thus, after micelles are injectedinto the bloodstream, they begin to equilibrate between the micellar,colloidal structure and individual surfactant molecules. Because of thechange in micellar structure and size, control over the release of drugswithin the micellar microcontainer cannot be maintained for longperiods. Typically drug is released over a period of hours and thisrelease is often inconsistent over this period. Thus, the thermodynamicequilibrium between surfactant and micelles may ultimately cause serioustoxicity problems due to potentially large fluctuations in drugconcentrations accompanied by the breakdown in micellar structure intosurfactant molecules. This dilution is particularly large after oral andintravenous administration and can cause unwanted precipitation ofhydrophobic drugs.

Thus, while micelles are frequently evaluated for use as drug deliverysystems, there are only a few products on the market that are consideredpractical. This is due to the eventual aggregation and/or precipitationof drugs resulting from equilibration of micelles back to the monomerand the solubilization capacity being too low to be of practical use.

Attempts have been made to design non-ionic surfactants such aspoly(ethylene oxide) containing molecules with improved solubilizationcharacteristics. An early approach involved the production of largemicellar systems. However, despite the increased micelle size,solubilization decreased with the longer hydrophobic chains. Thisdecrease was Attributed to deleterious changes in the poly(ethyleneoxide) chains nearest to the core, the main locus of solubilization formost drugs (Elworthy, P. and Patel, M. J. Pharm. Pharmacol. 198234:543).

Liu et al. (Polym. Preprint., 1997 38(2):582-583) report the synthesisof a single species of hyperbranched polymeric micelles forencapsulation of small hydrophobic organic molecules. This speciescontains no divalent amino acid moiety. Instead, this species comprisesa 1,1,1-tris(hydroxyphenyl) ethane moiety and an acylated mucic acidmoiety as the divalent dicarboxylic moiety. There remains a need forsuitable delivery systems for the administration of hydrophobicmolecules.

SUMMARY OF THE INVENTION

This need is met by the present invention. The present inventionprovides new hyperbranched polymer micelles that are useful forsolubilizing hydrophobic molecules in water thus greatly simplifying thepreparation of aqueous formulations for delivery of such molecules.

Therefore, according to one aspect of the present invention, a polymeris provided having a structure selected from:R(—O—R₁)_(x) and R(—NH—R₁)_(x),wherein R(—O—)_(x) is a polyol moiety and R(—NH—)_(x) is a polyaminemoiety, with x being between 2 and 10, inclusive, and each R₁independently has the structure:

-   wherein    is a divalent amino acid moiety with R₂ being a covalent bond or    having from 1 to 8 carbon atoms, and y and z are between 0 and 10,    inclusive, provided that y and z are not both 0;-   wherein    is a divalent dicarboxylic acid moiety in which R₃ is an alkylene or    cycloalkylene group containing from 1 to about 15 carbon atoms    substituted with a total of from 1 to about 10 hydroxyl groups, with    at least a portion of the hydroxyl groups being acylated with 3 to    24 carbon atom carboxylic acids; and-   wherein R₄ is a poly(alkylene oxide) having the structure:    R₅—(—R₆—O—)_(a)—R₆-Q-    with R₅ being selected from 1 to 40 carbon atom alkyl groups, —OH,    —OR₇—, NH₂, NHR₇, NR₇R₈, —C—OH, —C—OR₇, —C—O—C—R₇, —C—NH₂, C—NHR₇,    C—NR₇, C—NR₇R₈;-   R₆, R₇ and R₈ being independently selected from 2 to 40 carbon atom,    straight chain or branched alkylene groups;    -   Q being a divalent linkage moiety; and a being between 2 and        110, inclusive;-   with the proviso that when y is zero and R is a    1,1,1-tris(hydroxyphenyl) ethane moiety, the divalent dicarboxylic    moiety is not an acylated mucic acid moiety.

The polymers of the present invention encapsulate a wide variety ofhydrophobic molecules. The encapsulation is a physical encapsulation,and not a simple association of the hydrophobic molecule with thepolymer. According to a preferred embodiment of the present invention,upon formation of the encapsulated hydrophobic molecule, the polymer isrecovered and rinsed to remove any residue of non-encapsulatedhydrophobic molecules.

Therefore, according to another aspect of the present invention, ahydrophobic molecule encapsulated in a polymer is provided, wherein thepolymer has a structure selected from:R(—O—R₁)_(x), and R(—NH—R₁)_(x)wherein R(—O—)_(x) is a polyol moiety and R(—NH—)_(x) is a polyaminemoiety, with x being between 2 and 10, inclusive, and each R₁independently has the structure:

-   wherein    is a divalent amino acid moiety with R₂ being a covalent bond or    having from 1 to 8 carbon atoms, and y and z are between 0 and 10,    inclusive, provided that y and z are not both 0;-   wherein    is a divalent dicarboxylic acid moiety in which R₃ is an alkylene or    cycloalkylene group containing from 1 to about 15 carbon atoms    substituted with a total of from 1 to about 10 hydroxyl groups, with    at least a portion of the hydroxyl groups being acylated with 3 to    24 carbon atom carboxylic acids; and-   wherein R₄ is a poly(alkylene oxide) having the structure:    R₅—(—R₆—O—)_(a)—R₆-Q-    with R₅ being selected from 1 to 40 carbon atom alkyl groups, —OH,    —OR₇, —NH₂, —NHR₂, —NR₇R₈, —C—OH, —C—OR₇, —C—O—C—R₇, —C—NH₂, —C—NHR₇    and —C—NR₇R₈;-   R₆, R₇ and R₈ being independently selected from 2 to 40 carbon atom,    straight chain or branched alkylene groups;-   Q being a divalent linkage moiety; and    -   a being between 2 and 110, inclusive;    -   with the proviso that when y is zero and R is a        1,1,1-tris(hydroxyphenyl) ethane moiety, the divalent        dicarboxylic acid moiety is not a mucic acid moiety acylated        with carboxylic acids having less than six carbon atoms.

The present invention incorporates the discovery that acylation withcarboxylic acids of six carbon atoms or greater produces an unexpectedincrease in the affinity of the polymer interior for hydrophobicmolecules. The polymers of the present invention meet the need formicellar encapsulants for hydrophobic molecules that arethermodynamically stable in aqueous media. In a preferred embodiment,the hydrophobic molecules encapsulated by the polymers are hydrophobicmolecules with biological or pharmaceutical activity.

Furthermore, because the polymer encapsulates of the present inventionare thermodynamically stable in aqueous media, the present inventionalso includes aqueous solutions, suspensions and dispersions of polymerencapsulated hydrophobic molecules. Again, the hydrophobic moleculespreferably have biological or pharmaceutical activity.

The polymers of the present invention are ideal vehicles for thedelivery of hydrophobic molecules to a host via a variety ofadministration routes, including, but not limited to orally, topicallyor intravenously. Accordingly, the present invention also includesformulations comprising a polymer of the present invention encapsulatinghydrophobic molecules and an acceptable carrier or vehicle. In addition,the present invention includes methods of delivering a hydrophobicmolecule to a host by administering to the host a formulation comprisinga polymer of the present invention encapsulating hydrophobic moleculesand an acceptable carrier or vehicle. Acceptable vehicles for theformulations include, but are not limited to, aqueous solutions,suspensions, dispersions, emollients, lotions, creams, salves, balms andointments. The polymer encapsulated hydrophobic molecules can also beadministered in a solid form by way of a tablet or capsule, for example,to be dissolved in the digestive tract, and, consequently, in thebloodstream. By “host” it meant to include both plants and animalsincluding humans.

In a preferred embodiment, formulations are prepared for topicaladministration. The present invention incorporates the discovery thatthe encapsulation of hydrophobic molecules by the polymers of thepresent invention enhances transdermal delivery of the hydrophobicmolecule. In fact, absorption through the skin can be increased by afactor of up to 1000. Preferred formulations for transdermal deliveryinclude, but are not limited to aqueous solutions, suspensions,dispersions, aqueous gels and water-in-oil or oil-in-water emulsions.The formulations can also be applied directly to the skin as a lotion,cream or salve, or a transdermal delivery device such as a transdermalpatch can be employed, in which the polymer encapsulated hydrophobicmolecule is retained in the active agent reservoir of the patch. Inaddition to hydrophobic drugs, topical formulations are particularlyuseful in delivery of hydrophobic molecules including, but not limitedto, dyes, proteins, vitamins and fragrances, which are used routinely incosmetics, toiletries, fragrances, perfumes, skin care products andbeauty aids.

The polymer encapsulated hydrophobic molecules may be the only activemolecule in the topical formulation, or the formulation may contain thehydrophobic molecules stabilized in the formulation by other means, sothat the non-encapsulated hydrophobic molecule provides a “burst effect”upon initial delivery, followed by a sustained delivery of thepolymer-encapsulated molecule.

Therefore, according to another aspect of the present application, amethod for transdermal delivery to an animal in need thereof of ahydrophobic molecule having biological or pharmaceutical activity isprovided. An effective amount of a topical dosage form containing thehydrophobic molecule encapsulated by the polymer of the presentinvention and a pharmaceutically acceptable topical carrier, is appliedto the skin or mucosa of the animal. Preferred polymers according to thepresent invention hydrolyze into components known to be biocompatible,i.e., sugars, fatty acids, amino acids and poly(ethylene glycol). Thisalso results in low cytotoxicity of the polymer and its hydrolysisproducts. The poly(alkylene oxide) units enhance the immunogenicity ofthe encapsulate, enabling the hydrophobic molecules to evade the body'simmune system, thereby increasing the circulation time of thehydrophobic molecule. This allows for effective treatment with reducedquantities of the hydrophobic molecule, which, together with theenhanced immunogenicity, prevents or reduces the severity and incidenceof toxic side effects of the hydrophobic molecules.

The polymeric micelles have also been found to stabilize liposomes andother lipid-based structures for extended periods of time so thatprecipitation is prevented and the optical transparency of formulationsis maintained. Therefore, another aspect of this invention relates tothe use of these polymeric micelles in the stabilization of formulationscomprising liposomes or other lipid based structures.

The above and other objects, features and advantages of the presentinvention will become clear from the following description of thepreferred embodiment.

DETAILED DESCRIPTION OF THE INVENTION

The polymers of the present invention are prepared by coupling aplurality of hydrophobic moieties to the hydroxyl groups of a polyolcore. The resulting polymer is then made water-soluble by attaching apoly(alkylene oxide) to the end of each hydrophobic moiety. Polyols thatare suitable for use as the polymer core are nearly limitless. Aliphaticpolyols having from 1 to 10 carbon atoms and from 1 to 10 hydroxylgroups may be used, including ethylene glycol, alkane diols, alkylglycols, alkylidene alkyl diols, alkyl cycloalkane diols,1,5-decalindiol, 4,8-bis(hydroxymethyl)tricyclodecane, cycloalkylidenediols, dihydroxyalkanes, trihydroxyalkanes, and the like. Cycloaliphaticpolyols may also be employed, including straight chained or closed-ringsugars and sugar alcohols, such as mannitol, sorbitol, inositol,xylitol, quebrachitol, threitol, arabitol, erythritol, adonitol,dulcitol, facose, ribose, arabinose, xylose, lyxose, rhamnose,galactose, glucose, fructose, sorbose, mannose, pyranose, altrose,talose, tagitose, pyranosides, sucrose, lactose, maltose, and the like.More examples of aliphatic polyols include derivatives ofglyceraldehyde, glucose, ribose, mannose, galactose, and relatedstereoisomers.

Aromatic polyols are preferred because of their hydrophobicity. Amongthe suitable aromatic polyols are 1,1,1-tris(4′-hydroxyphenyl)alkanes,such as 1,1,1-tris(4-hydroxyphenyl)ethane, (1,3-adamantanediyl)diphenol,2,6-bis(hydroxyalkyl)cresols,2,2′alkylene-bis(G-t-butyl-4-alkylphenols),2,2′-alkylene-bis(t-butylphenols), catechol, alkylcatechols, pyrogallol,fluoroglycinol, 1,2,4-benzenetriol, resorcinol, alkylresorcinols,dialkylresorcinols, orcinol monohydrate, olivetol, hydroquinone,alkylhydroquinones, 1,1-bi-2-naphthol, phenyl hydroquinones,dihydroxynaphthalenes, 4,41-(9-fluorenylidene)-diphenol, anthrarobin,dithranol, bis(hydroxyphenyl)methane biphenols, dialkylstilbesterols,bis(hydroxyphenyl) alkanes, bisphenol-A and derivatives thereof,meso-hexesterol, nordihydroguaiaretic acid, calixarenes and derivativesthereof, tannic acid, and the like.

Other core polyols that may be used include cyclic crown ethers,cyclodextrines, dextrines and other carbohydrates such as starches andamylose. Alkyl groups may be straight-chained or branched, and maycontain from 1 to 10 carbon atoms.

Hydrophobic moieties are coupled to two or more of the core polyolhydroxyl groups. Preferably, all of the hydroxyl groups of the corepolyol are coupled to a hydrophobic moiety.

According to one embodiment of the present invention, the hydrophobicmoiety is a dicarboxylic acid moiety containing from 1 to about 10carbon atoms and substituted with from 1 to about 10 hydroxyl groups,wherein at least a portion of the hydroxyl groups are acylated with 3 to24 carbon atom carboxylic acids. One carboxylic acid group of thedicarboxylic acid is coupled to a hydroxyl group of the core polyol byway of an ester linkage, while the other carboxylic acid group remainsfree for coupling to the poly(alkylene oxide). The dicarboxylic acid maybe a straight chained or branched, aliphatic or cycloaliphatic,dicarboxylic acid. Suitable aliphatic dicarboxylic acids include mucicacid, malic acid, citromalic acid, alkylmalic acid, hydroxy derivativesof glutaric acid, and alkyl glutaric acids, tartaric acid, citric acid,hydroxy derivatives of fumaric acid, and the like. Alkyl groups may bestraight-chained or branched and may contain from 1 to 10 carbon atoms.The cycloaliphatic dicarboxylic acids include dicarboxylic acidderivatives of sugar alcohols.

The carboxylic acids acylating the hydroxyl groups of the dicarboxylicacids preferably contain from 6 to 24 carbon atoms. Preferably, everyhydroxyl group of a dicarboxylic acid is acylated with a carboxylicacid.

The polyol coupled to two or more acylated dicarboxylic acid branchesforms the hydrophobic core of the polymer of the present invention.According to another embodiment of the present invention, the volume ofthe cavity formed by the hydrophobic core of the polymer may beincreased by inserting an amino acid or peptide linkage between the corepolyol and each hydrophobic moiety. That is, a linkage as small as oneamino acid up to the size of an oligopeptide containing 10 amino acidresidues may be attached to each core polyol hydroxyl group or polyamineamino group, with the hydrophobic moiety being coupled to the end of theamino acid or peptide opposite the core polyol hydroxyl group orpolyamine amino group.

The carboxylic acid terminus of an amino acid or peptide is coupled to ahydroxyl group of the core polyol by an ester linkage or an amino groupof a core polyamine by an amide linkage. A carboxylic acid group of theacylated dicarboxylic acid is then coupled to the amine terminus of theamino acid or peptide by an amide linkage. The other carboxylic acidgroup again remains free for coupling to a poly(alkylene oxide). Thenumber of amino acids employed in each peptide linkage should not be sogreat as to render the entire polymer water-insoluble. A peptide linkagecontaining from 3 to 6 amino acids is preferred. Preferred amino acidsinclude lysine, serine, threonine, cysteine, tyrosine, aspartic acid,glutamic acid and arginine. Like the other components of the polymers ofthe present invention, the amino acid linkages also hydrolyze to formbiocompatible degradation products.

The free carboxylic acids on the end of each hydrophobic branch on thepolyol core are then coupled to a poly(alkylene oxide). Thepoly(alkylene oxides) are preferably coupled to the free carboxylicacids by either ester or amide linkages. The alkylene oxide unitscontain from 2 to 4 carbon atoms and may be straight, chained orbranched. Poly(ethylene glycol) (PEG) is preferred. Alkoxy-terminatedpoly(alkylene oxides) are preferred, with methoxy-terminatedpoly(alkylene oxides) being more preferred.

The poly(alkylene oxide) preferably has between about 2 and about 110repeating units. A poly(alkylene oxide) having between about 50 andabout 110 repeating units is more preferred.

The polymers of the present invention are prepared by first acylatingthe hydroxyl substituted dicarboxylic acid. The dicarboxylic acid isreacted with a stoichiometric excess of the appropriate acyl chloride inthe presence of a catalyst, if needed, such as ZnCl₂ with heating, up tothe reflux temperature of the reaction mixture. Those of ordinary skillin the art will understand that the appropriate acyl chloride will havefrom about 2 to about 24, and preferably from about 6 to about 24,carbon atoms.

The reaction continues until substantially complete, approximately 5hours, after which the reaction product is extracted into an ether suchas diethyl ether, followed by washing of the ether fraction with water,drying and evaporation. The resulting crude product is the purified byrecrystallization.

The acylated dicarboxylic acid is then coupled to a core polyol by meansof a carbodiimide-mediated coupling reaction. The core polyol and astoichiometric excess of the acylated dicarboxylic acid are dissolved ina common solvent, such as an ether, for example, diethyl ether.

Carbodiimide-mediated coupling reactions are disclosed in Bodanszky,Practice of Peptide Synthesis, (Springer-Verlag, New York, 1984) at page145. A quantity of a solution providing a molar equivalent of acarbodiimide and N,N-dimethylaminopyridine (DMAP) for each polyolhydroxyl group, dissolved in a common solvent such as methylenechloride, is added to the reaction mixture. The reaction proceedsrapidly to completion, after which the urea side-product correspondingto the carbodiimide is removed by suction filtration. The filtratesolution is then washed and dried, and the reaction solvent is thenevaporated to recover the crude reaction product. The crude product isthen purified, for example, by flash chromatography.

Carbodiimides suitable for use with the present invention includedicyclohexylcarbodiimide (DCC) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,1-alkyl-3-(3-dimethylaminopropyl)carbodiimide(alkyl=isopropyl,cyclochexyl), 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl))carbodiimide,1-cyclohexyl-3-(4-diethylaminocyclohexyl)carbodiimide,1-cyclohexyl-3-(diethylaminoethyl)carbodiimide, 1,3-di-(4-diethylaminocyclohexyl)carbodiimide,1-alkyl-3-(3-morpholinyl-(4-propyl))carbodiimide (alkyl=methyl, ethyl),1-benzyl-3-(3-dimethylamino-(N)-propyl)carbodiimide, and1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. In each case, thecarbodiimide is used as the free base or a salt (HCl, methiodide,metho-p-toluenesulfonate, and the like). The preferred carbodiimide isDCC.

Carbodiimide-mediated coupling reactions are also used to create anamino acid or peptide linkage between the core polyol hydroxyl groups orcore polyamine amino groups and the acylated dicarboxylic acids. Thecoupling reaction is first performed between the core polyol orpolyamine and the amino acid or peptide, after which the amino acid orpeptide-branched polyol or polyamine is then reacted with the acylateddicarboxylic acid in another carbodiimide-mediated coupling reaction.

The poly(alkylene oxide) chains are then attached by reacting thesubstituted core polyol or polyamine with an activated poly(alkyleneoxide) in another carbodiimide-mediated coupling reaction. To attach thepoly(alkylene oxide) chains by way of an amide linkage, poly(alkyleneoxide) amines are employed. For an ester linkage, a poly(alkylene oxide)is employed. For an anhydride linkage, a poly(alkylene oxide) carboxylicacid is employed. Other linkages represented by Q in the above formulasthat are suitable for use with the present invention are well known tothose skilled in the pegylation art and require no further description.

A reaction mixture of the substituted core polyol and the activatedpoly(alkylene oxide) in a common solvent such as methylene chloride isformed. A quantity of a solution providing a molar equivalent ofcarbodiimide and DMAP for each substituted hydroxyl group of the corepolyol, dissolved in a common solvent such as methylene chloride, isthen added to the reaction mixture. The reaction mixture is maintainedat room temperature with stirring for at least 12 hours, after which itis evaporated to dryness, followed by purification, for example byrecrystallization, followed by flash chromatography.

The polymers of the present invention have a number average molecularweight between about 1,000 and about 100,000 daltons, measured by GelPermeation Chromatography relative to polystyrene standards. Molecularweights between about 2,500 and about 25,000 daltons are preferred.

The resulting polymers may be used for essentially any application inwhich conventional micelles are employed. Examples include drugsolubilization, fragrance encapsulation, passive targeting for drugdelivery, waste water treatment, enhanced capillary electrophoresisactivation, and induction of protein crystallization. Alkali metalcations may also be encapsulated by the polymer to form solvent-freepolymer-salt complex solid electrolytes.

Polymeric micelles, unlike conventional micelles, do not have propertiesthat are dependent on concentration or temperature. Thus, polymericmicelles do not have a CMC. Typical micelles can only encapsulatehydrophobic moieties when above the CMC and within a specifictemperature range. In contrast, the polymeric systems of the presentinvention are useful at concentrations typically well below that ofmicelles in current use. For example, effective liposome stabilizationhas been achieved at 10⁻¹⁰ M. Use of the polymeric micelles toencapsulate these hydrophobic molecules thus provides multipleadvantages. Specifically, the polymeric micelles act as dispersingagents for hydrophobic molecules such as dyes, fragrances, proteins,vitamins and biologically active molecules thus providing a more evendistribution as compared to currently available technologies such asdetergents, micelles, surfactants, fatty acids, lipids, amphiphiles andcolloids. The polymeric micelles also have the ability to stabilizeliposomes, emulsions, solutions, suspensions, dispersions, aqueous gels,water-in-oil or oil-in-water emulsions, and microemulsions for extendedperiods of time at ambient, refrigerated and elevated temperatures.Further, the spherical architecture of the polymeric micelles as opposedto agents with a linear architecture such as lipids and amphiphiles, isbelieved to increase lubricating ability by decreasing solutionviscosity. Polymeric micelles of the present invention also have theability to encapsulate extremely hydrophobic molecules such as lidocaineand naphthalenes. Because the hydrophobic moieties are encapsulated,they become completely water-soluble thus enhancing their absorptionupon topical application. Aqueous solutions of the polymeric micelleencapsulated hydrophobic molecules are transparent and do not requireaddition of other solubilizing agents. Finally, the polymeric micelleshave been demonstrated to be non-toxic to cells such as fibroblasts andare believed to be completely biocompatible.

According to one embodiment of the present invention, hydrophobicmolecules are encapsulated by dissolving the hydrophobic molecules andthe polymer in a common solvent, such as methylene chloride. The solventis then removed, for example, by rotoevaporation. The resulting solid isthen washed thoroughly with a non-polar solvent such as hexane, toremove any residual non-encapsulated hydrophobic materials. The washedsolid is then thoroughly dried, preferably under vacuum, to completelyremove any adsorbed solvent, and to obtain the essentially purepolymer-encapsulated hydrophobic material.

According to an alternative embodiment, the polymer of the presentinvention is dissolved in water, and an excess quantity of thehydrophobic material is added to the aqueous solution, with stirring.After allowing the hydrophobic material sufficient contact with theaqueous polymer solution, the excess hydrophobic material is permittedto separate from the aqueous solution, after which it is removed. Thepolymer-encapsulated material may then be kept in this aqueous solution,or the aqueous solution may be concentrated, or the polymer encapsulatemay be recovered in dry form by evaporating the water.

When the water is evaporated, the dried polymer may be subjected tonon-polar solvent rinsing to remove any residual hydrophobic materialand further evaporation to remove any residual adsorbed non-polarsolvent.

The present invention contemplates the use of polymer-encapsulatedhydrophobic molecules at concentrations as high as 1 M and greater, upto 10⁶ M. At the same time, another advantage of the present inventionis the thermodynamic stability of the polymers, which permit theformation of low concentration stable aqueous solutions of the polymerencapsulates, far below the CMCs of conventional surfactants. Stableaqueous solutions as low as 10⁻¹⁰ M have been obtained, although, atpresent, concentrations of 10⁻⁸ and greater are expected to have thegreatest commercial utility. The polymers of the present invention arebelieved to form stable aqueous encapsulate solutions below thepresently available limits of detection, i.e., below 10⁻¹⁰ M.

In a preferred embodiment of the present invention, the polymers areused to solubilize hydrophobic molecules with biological orpharmaceutical activity for drug delivery.

Pharmaceutical dosage forms of polymer-encapsulated hydrophobicmolecules having biological or pharmaceutical activity may be formulatedusing physiologically acceptable carriers, excipients, stabilizers andthe like, and may be provided in sustained release or timed releaseformulation. Acceptable carriers, excipients and diluents fortherapeutic use are well known in the pharmaceutical field, and aredescribed, for example, in Remington's Pharmaceutical Science (A. R.Gennaro Edit., Mack Publishing Co., 1985). Such materials are non-toxicto the recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, acetate and other organicacid salts, antioxidants such as ascorbic acid, low molecular weight(less than about 10 residues) peptides such as polyarginine, proteins,such as serum albumin, gelatin and immunoglobulins, hydrophilic polymerssuch as poly(vinyl pyrrolidinone), amino acids such as glycine, glutamicacid, aspartic acid and arginine, monosaccharides, disaccharides, andother carbohydrates, including cellulose and its derivatives, glucose,mannose and dextrines, chelating agents such as EDTA, sugar alcoholssuch as mannitol and sorbitol, and conventional cationic and nonionicsurfactants such as TWEEN, PULRONICS, and PEG. Dosage formulations to beused for therapeutic administration must be sterile. Sterility isreadily accomplished by filtration through sterile membranes, or byother conventional methods such as irradiation or treatment with gasesor heat. The pH of the dosage formulations of this invention typicallywill be between 3 and 11, and more preferably from 5 to 9. Hosts in needof treatment (typically mammalian) using the dosage formulations of thisinvention can be administered dosages that will provide optimalefficacy. The dose and method of administration will vary from subjectto subject and be dependent upon such factors as the type of host beingtreated, and in the case of animals, its sex, weight, diet, concurrentmedication, overall clinical condition, the particular hydrophobiccompounds employed, the specific use for which these compounds areemployed, and other factors which those skilled in the arts willrecognize.

Therapeutically effective dosages may be determined by either in vitroor in vivo methods. For each particular dosage form of the presentinvention, individual determinations may be made to determine theoptimal dosage required. The range of therapeutically effective dosageswill naturally be influenced by the route of administration, thetherapeutic objectives, and the condition of the host. For the varioussuitable routes of administration, the absorption efficiency must beindividually determined for each hydrophobic compound by methods wellknown in pharmacology. Accordingly, it may be necessary for thetherapist to titer the dosage and modify the route of administration asrequired to obtain the optimal therapeutic effect. The determination ofeffective dosage levels, that is, the dosage levels necessary to achievethe desired result, will be within the ambit of one skilled in the art.

Typically, applications of compound are commenced at lower dosagelevels, with dosage levels being increased until the desired effect isachieved.

A typical dosage might range from about 0.001 mg to about 1,000 mg ofhydrophobic material, per kg of patient weight. Preferred dosages rangefrom about 0.01 mg/kg to about 100 mg/kg, and more preferably from about0.10 mg/kg to about 20 mg/kg. Advantageously, the dosage forms of thisinvention may administered several times daily, and other dosageregimens may also be useful.

The dosage formulations of this invention may be prepared for storageunder conditions suitable for the preservation of the biological orpharmaceutical activity of the hydrophobic material, as well as formaintaining the integrity of the polymer, and are typically suitable forstorage in ambient or refrigerated temperatures. The polymerencapsulates of the present invention may be formulated foradministration orally, subcutaneously, intramuscularly, intravenously,colonically, rectally, nasally or intraperitonially, employing a varietyof dosage forms such as solutions, tablets, capsules, gelcaps,suppositories, implanted pellets or small cylinders, aerosols andtopical formulations such as lotions, ointments, drops and dermalpatches. The dosage formulations of this invention are suitable forapplications where localized drug delivery is desired, as well as insituations where a systemic delivery is desired.

The dosage formulations of this invention may desirably furtherincorporate agents to facilitate the systemic delivery of thehydrophobic material having biological or pharmaceutical activity to thedesired target. The hydrophobic materials to be delivered may, in thisfashion, be incorporated with antibodies, antibody fragments, growthfactors, hormones, or other targeting moieties, to which the drugmolecules are coupled. The present invention also contemplates the useof peptide linkages between the core polyol and the acylateddicarboxylic acids that are selected for cleavage by proteolyticenzymes, resulting in degradation of the polymer and release of theencapsulated hydrophobic material. The release of the hydrophobicmaterial may thus be targeted by selecting a peptide sequencecorresponding to a proteolytic enzyme unique to the target site, or byco-administering a proteolytic enzyme corresponding to the peptidesequence at the target site for the hydrophobic material.

However, one advantage of the polymers of the present invention is thatpolymer degradation is not a prerequisite for release of the hydrophobicmaterial.

A particular class of hydrophobic molecules having biological activitythat are suitable for use with the present invention are inter-cellularregulators and mediators such as interferons, growth factors, hormones,and the like. The polymers of the present invention are contemplated tobe particularly effective for the efficient administration ofinterferons, which has proven to be problematic because of interferon'swater-insolubility. As noted above, the topical dosage forms of thepresent invention exhibit an unexpectedly accelerated rate oftransdermal delivery attributable to the encapsulation of thehydrophobic material by the polymers of the present invention. Thus, thepolymer-encapsulated hydrophobic material having biological orpharmaceutical activity may be prepared as topical dosage forms such aslotions, gels, salves, creams, balms, ointments and the like. Thesecompositions may be in the form of aqueous solutions, or in the form ofoil-in-water or water-in-oil emulsions. The formulations are essentiallyconventional, containing well-known additives, and are prepared usingart-recognized techniques.

Topical dosage forms may also be prepared by incorporating the polymerencapsulate into the reservoir of a transdermal drug delivery device.Transdermal administration systems, or “patches”, are well-known in theart. Occlusive transdermal patches for the administration of an activeagent to the skin or mucosa are described in U.S. Pat. Nos. 4,573,966;4,597,961 and 4,839,164, the disclosures of which are incorporatedherein by reference.

Essentially any device capable of delivering an active agenttransdermally may be employed to transdermally deliver the polymerencapsulate of the present invention.

As noted above, the polymer encapsulates are particularly well suitedfor efficient delivery through the dermis. By “efficient” it is meantthat a high level of the encapsulate is evenly delivered or transportedover time.

However, practically any hydrophobic therapeutic agent otherwisesuitable for the practice of this invention may be employed for avariety of applications. The polymers of the present invention can alsobe used as thickening agents, lubricants, detergents surfactants,plasticizers and anti-fouling agents. The polymers may be used as anemulsifying, dispersing or stabilizing agent for dyes, cosmetics,pigment and pharmaceutical products. The polymers are particularlyuseful as an emulsifying, dispersing or stabilizing agent in the dyeingof textiles and for encapsulating dyes for cosmetics. The polymers areuseful as lubricants and encapsulants for cosmetics, textiles, leathersand perfumes, and are particularly useful as a thickening agent forpaints. The polymers may also be employed as an emulsifying, dispersingor stabilizing agent for components of photographic compositions anddevelopers.

Thus, in addition to biologically or pharmaceutically active hydrophobicmolecules, other hydrophobic molecules that may be encapsulated by thepolymers of the present invention include insecticides, pesticides,herbicides, antiseptics, food additives, fragrances, dyes, diagnosticaids, and the like.

In a preferred embodiment, the hyperbranched polymeric micelles are usedas a replacement for detergents, micelles, surfactant, fatty acids,lipids, amphiphiles, colloids and other delivery agents currently usedin cosmetics, toiletries, fragrances, perfumes, skin care products andbeauty aids. In this embodiment, the hyperbranched polymeric micellesare used to encapsulate hydrophobic molecules such as dyes, proteins,vitamins, biologically active molecules and fragrances which are thenincorporated into aqueous solutions, suspension, dispersion, emollients,lotions, creams, salves, balms and ointment for topical delivery ofthese hydrophobic molecules.

Examples of hydrophobic molecules that may be encapsulated by thepolymers of the present invention include, but are not limited to:abietic acid, aceglatone, acenaphthene, acenocournarol, acetohexamide,acetomeroctol, acetoxolone, acetyldigitoxins, acetylene dibromide,acetylene dichloride, acetylsalicylic acid, alantolactone, aldrin,alexitol sodium, allethrin, allylestrenol, allylsulfide, alprazolam,aluminum bis(acetylsalicylate), ambucetamide, aminochlothenoxazin,aminoglutethimide, amyl, chloride, androstenediol, anethole trithone,anilazine, anthralin, Antimycin A, aplasmomycin, arsenoacetic acid,asiaticoside, asternizole, aurodox, aurothioglycanide, 8-azaguanine,azobenzene; baicalein, Balsam Peru, Balsam Tolu, barban, baxtrobin,bendazac, bendazol, bendroflumethiazide, benomyl, benzathine,benzestrol, benzodepa, benzoxiquinone, benzphetamine, benzthiazide,benzyl benzoate, benzyl cinnamate, bibrocathol, bifenox, binapacryl,bioresmethrin, bisabolol, bisacodyl, bis(chlorophenoxy)methane, bismuthiodosubgallate, bismuth subgallate, bismuth tannate, Bisphenol A,bithionol, bornyl, bromoisovalerate, bornyl chloride, bornylisovalerate, bornyl salicylate, brodifacoum, bromethalin,broxyquinoline, bufexamac, butamirate, butethal, buthiobate, butlatedhydroxyanisble, butylated hydroxytoluene; calcium iodostearate, calciumsaccharate, calcium stearate, capobenic acid, captan, carbamazepine,carbocloral, carbophenothin, carboquone, carotene, carvacrol,cephaeline, cephalin, chaulmoogric acid, chenodiol, chitin, chlordane,chlorfenac, chlorfenethol, chlorothalonil, chlorotrianisene,chlorprothixene, chlorquinaldol, chromonar, cilostazol, cinchonidine,citral, clinofibrate, clofaziminc, clofibrate, cloflucarban, clonitrate,clopidol, clorindione, cloxazolam, coroxon, corticosterone, cournachlor,coumaphos, coumithoate cresyl acetate, crimidine, crufomate, cuprobam,cyamemazine, cyclandelate, cyclarbamate cymarin, cypermethril; dapsone,defosfamide, deltamethrin, deoxycorticocosterone acetate,desoximetasone, dextromoramide, diacetazoto, dialifor, diathymosulfone,decapthon, dichlofluani, dichlorophen, dichlorphenamide, dicofol,dicryl, dicumarol, dienestrol, diethylstilbestrol, difenamizole,dihydrocodeinone enol acetate, dihydroergotamine, dihydromorphine,dihydrotachysterol, dimestrol, dimethisterone, dioxathion, diphenane,N-(1,2-diphenylethyl)nicotinamide, dipyrocetyl, disulfamide, dithianone,doxenitoin, drazoxolon, durapatite, edifenphos, emodin, enfenamic acid,erbon, ergocorninine, erythrityl tetranitrate, erythromycin stearate,estriol, ethaverine, ethisterone, ethyl biscournacetate,ethylhydrocupreine, ethyl menthane carboxamide, eugenol, euprocin,exalamide; febarbamate, fenalamide, fenbendazole, fenipentol,fenitrothion, fenofibrate, fenquizone, fenthion, feprazone, flilpin,filixic acid, floctafenine, fluanisone, flumequine, fluocortin butyl,fluoxymesterone, flurothyl, flutazolam, fumagillin,5-furftiryl-5-isopropylbarbituric acid, fusaftmgine, glafenine,glucagon, glutethimide, glybuthiazole, griseofulvin, guaiacol carbonate,guaiacol phosphate, halcinonide, hematoporphyrin, hexachlorophene,hexestrol, hexetidine, hexobarbital, hydrochlorothiazide, hydrocodone,ibuproxam, idebenone, indomethacin, inositol niacinate, iobenzamic acid,iocetamic acid, iodipamide, iomeglamic acid, ipodate, isometheptene,isonoxin, 2-isovalerylindane-1,3-dione; josamycin, 11-ketoprogesterone,laurocapram, 3-O-lauroylpyridoxol diacetate, lidocaine, lindane,linolenic acid, liothyronine, lucensomycin, mancozeb, mandelic acid,isoamyl ester, mazindol, mebendazole, mebhydroline, mebiquine,melarsoprol, melphalan, menadione, menthyl valerate, mephenoxalone,mephentermine, mephenyloin, meprylcaine, mestanolone, mestranol,mesulfen, metergoline, methallatal, methandriol, methaqualone,methylcholanthrene, methylphenidate, 17-methyltestosterone,metipranolol, minaprine, myoral, naftalofos, naftopidil, naphthalene,2-naphthyl lactate, 2-(2-naphthyloxy)ethanol, naphthyl salicylate,naproxen, nealbarbital, nemadectin, niclosamide, nicoclonate,nicomorphine, nifuroquine, nifuroxazide, nitracrine, nitromersol,nogalamycin, nordazepam, norethandrolone, norgestrienone; octaverine,oleandrin, oleic acid, oxazeparn, oxazolam, oxeladin, oxwthazaine,oxycodone, oxymesterone, oxyphenistan acetate, paraherquamide,parathion, pemoline, pentaerythritol tetranitrate, pentylphenol,perphenazine, phencarbamide, pheniramine, 2-phenyl-6-chlorophenol,phenthnethylbarbituric acid, phenytoin, phosalone,O-phthalylsulfathiazole, phylloquinone, picadex, pifamine, piketopfen,piprozolin, pirozadil, plafibride, plaunotol, polaprezinc, polythiazide,probenecid, progesterone, promegestone, propanidid, propargite, propham,proquazone, protionamide, pyrimethamine, pyrimithate, pyrvinium pamoate;quercetin, quinbolone, quizalofo-ethyl, rafoxanide, rescinnamine,rociverine, ronnel; salen, scarlet red, siccanin, simazine, simetride,sobuzoxane, solan, spironolactone, squalene, stanolone, sucralfate,sulfabenz, sulfaguanole, sulfasalazine, sulfoxide, sulpiride,suxibuzone, talbutal, terguide, testosterone, tetrabromocresol,tetrandrine, thiacetazone, thiocolchicine, thioctic acid, thioquinox,thioridazine, thiram, thymyl N-isoamylcarbamate, tioxidazole, tioxolone,tocopherol, tolciclate, tolnaftate, triclosan, triflusal, triparanol;ursolic acid, valinomycin, verapamil, vinblastine, vitamin A, vitamin D,vitamin E, xenbucin, xylazine, zaltoprofen, and zearalenone.

The following non-limiting examples set forth hereinbelow illustratecertain aspects of the invention. All parts and percentages are byweight unless otherwise noted and all temperatures are in degreesCelsius. All PEGs were obtained from Shearwater Polymers (Birmingham,Ala.) and used without further purification. All other chemicals wereobtained from Aldrich (Milwaukee, Wis.) and used without furtherpurification. Analytical grade solvents were used for all the reactions.Methylene chloride, tetrahydrofuran (THF), triethylamine (TEA) anddimethylsulfoxide (DMSO) were distilled. Nuclear magnetic resonancespectroscopy (¹H NMR, ¹³C NMR), infrared spectroscopy (IR), massspectrometry (MS), gel permeation chromatography (GPQ and elementalanalysis were used for physicochemical characterization. Fordifferential scanning calorimetry (DSC) measurements, samples wereheated under dry nitrogen gas. Data were collected at heating andcooling rates of 10° C./minute with a two cycle minimum. Forthermogravimetric analysis (TGA), samples were also heated under drynitrogen gas. Data were collected at a heating rate of 20° C./minute.Molecular weights were determined by GPC relative to narrow molecularweight polystyrene standards.

EXAMPLES Examples 1-3 Acylation of Mucic Acid Example 1 Mucic AcidPropyl Ester

To a neat mixture of mucic acid (4.2 g, 20 mmol) and propionyl chloride(18 ml, 200 mmol) was added ZnCl₂ (0.28 g, 2.0 mmol). The reactionmixture was heated at reflux temperature for three hours. After cooling,diethyl ether (20 ml) was added to the reaction mixture and the solutionpoured onto ice chips (approximately 100 g) with stirring. Additionaldiethyl ether (80 ml) was added to the mixture and stirring continuedfor 30 minutes more. The ether portion was separated, washed with waterto a neutral pH, dried over anhydrous Na₂SO₄ and evaporated to dryness.The crude product was purified by recrystallization from a cosolventsystem of diethyl ether and methylene chloride, collected by vacuumfiltration, washed by ice cold methylene chloride and dried at 105° C.(12 hours) to constant weight. A white solid having a T_(m) of 196° C.was obtained at a 56% yield.

Example 2 Mucic Acid Hexyl Ester

Mucic acid hexyl ester was prepared as in Example 1, substitutingcaproyl chloride for propionyl chloride. A white solid having a T_(m) of171° C. was obtained at a yield of 68%.

Example 3 Mucic Acid Lauryl Ester

Mucic acid lauryl ester was prepared as in Example 1, substitutinglauryl chloride for propionyl chloride. A white solid having a T_(m) of145° C. was obtained at a yield of 65%.

Examples 4-6 Preparation of Polymer Core Example 4 Propyl Ester

The mucic acid propyl ester of Example 1 (6.0 mmol) and1,1,1-tris(4′-hydroxyphenyl) ethane (0.51 g, 1.7 mmol) were dissolved inanhydrous ethyl ether (150 ml). To the reaction mixture, a solution ofDCC (1.2 g, 6.0 mmol) and DMAP (0.74 g, 6.0 mmol) in 25 ml methylenechloride was added dropwise. After 15 minutes, the DCC side-product(dicyclohexylurea) was removed by suction filtration. The filtrate waswashed with 20 ml portions of 0.1 N HCl (2×) and brine (4×), dried overanhydrous Na₂SO₄, and evaporated to dryness. The crude product waspurified by flash chromatography using ethyl ether:methanol:acetic acid(90:5:5) as eluent. A white solid having a T_(m) of 158° C. was obtainedat 58% yield.

Example 5 Hexyl Ester

The hexyl ester core molecule was prepared according to the method ofExample 4, substituting the mucic acid hexyl ester of Example 2 for themucic acid propyl ester. A white solid having a T_(m) of 147° C. wasobtained at 36% yield.

Example 6 Lauryl Ester

The lauryl ester core molecule was prepared according to the method ofExample 4, substituting the mucic acid lauryl ester of Example 3 for themucic acid propyl ester. A white solid having a T_(m) of 136° C. wasobtained at yield of 33%.

Examples 7-11 Preparation of Final Polymers Example 7 Mucic Acid HexylEster Core Polymer with Triethylene Glycol (TEG) Branches

To a mixture of the core molecule of Example 5 (0.106 mmol) andmethoxyterminated triethylene glycol amine (0.351 mmol) in 20 ml ofmethylene chloride at room temperature, DCC (0.351 mmol) and DMAP (0.351mmol) in 2 ml methylene chloride was added dropwise. After three days,the reaction mixture was evaporated to dryness, the residue dissolvedinto 20 ml methanol, and the crude product precipitated from 400 mlpetroleum ether at room temperature. The crude product was firstpurified by flash chromatography using ethyl ether:methanol:acetic acid(90:5:5) as eluent, then further purified by repetitive precipitationusing methylene chloride as solvent and diethyl ether/petroleum ether asnon-solvent. The ratio between methylene chloride and ethers wasprogressively changed. A white solvent was obtained having a T_(m) of31° C., a T_(d) of 220° C. and M_(w) of 2,400 daltons at a yield of 15%.

Example 8 Mucic Acid Hexyl Ester Core Polymer with PEG 2000 Branches

A mucic acid hexyl ester core polymer with PEG 2000 branches wasprepared according to the method of Example 7, substitutingmethoxy-terminated poly(ethylene glycol) amine (H₂N-m-PEG 2000,M_(w)=2000) for the methoxy-terminated triethylene glycol amine ofExample 7. A white solid was obtained having a T_(m) of 54° C. and aM_(w) of 9,400 daltons at a yield of 25%.

Example 9 Mucic Acid Hexyl Ester Core Polymer with PEG 5000 Branches

A mucic acid hexyl ester core polymer with PEG 5000 branches wasprepared according to the method of Example 7, substitutingmethoxy-terminated poly(ethylene glycol) amine (H₂N-PEG 5000, MW=5000)for the methoxy-terminated triethylene glycol amine of Example 7. Awhite solid having a T_(m) of 61° C. and a M_(w) of 17,800 daltons wasobtained at 17% yield.

Example 10 Mucic Acid Propyl Ester Core Polymer with PEG 5000 Branches

Mucic acid propyl ester core polymer with PEG 5000 branches was preparedaccording to the method of Example 9, substituting the mucic acid propylester core polymer of Example 4 for the mucic acid hexyl ester corepolymer. A white solid was obtained having a T_(m) of 62° C. and a M_(w)of 17,000 daltons at 30% yield.

Example 11 Mucic Acid Lauryl Ester Core Polymer with PEG 5000 Branches

Mucic acid lauryl ester core polymer with PEG 5000 branches was preparedaccording to the method of Example 9, substituting the mucic acid laurylester core polymer of Example 6 for the mucic acid hexyl ester corepolymer. A white solid was obtained having a T_(m) of 60° C. and a M_(w)of 19,100 daltons at a yield of 45%.

For the polymers of Examples 8-11, TGA showed two stages ofdecomposition. The first stage corresponded to cleavage of the corestructures from the ethylene oxide chains (about 200° C.) with theappropriate weight loss, and the second stage corresponded todecomposition of the ethylene oxide chain.

Example 12 Encapsulation Studies

Lidocaine (50 mg) and the polymer of Example 9 (50 mg) were dissolved in2.0 ml methylene chloride. The solution was evaporated to dryness andthe solid residue extensively washed with hexane until lidocaine was nolonger detected in the washings. The solid was dried under vacuum at 25°C. for about 2 hours. A portion (5.0 mg) of this solid was dissolvedinto methanol (1.0 ml) to release the entrapped lidocaine, and thelidocaine concentration was quantified by high pressure liquidchromatography (HPLC) according to a calibration curve generated from aseries of standard solutions ranging from 0.005 to 0.5 mg/ml lidocaine.The linearity of the curve indicated a direct, proportional relationshipbetween absorbance and lidocaine concentration. Using the equation ofthe lidocaine calibration curve, the amount of lidocaine entrapped inthe micelle core was determined. PEG with a molecular weight of 5,000daltons was used as the HPLC control.

Encapsulation number was defined as the amount of molecules that can beentrapped within the polymeric micelles. The values for the polymers ofExample 9, 10 and 11 were 1.0, 0.7 and 1.6 weight %, respectively. Theencapsulation number increased as the hydrophobicity of the polymerinterior increased.

The PEG arms of the polymers of the present invention thus form ahydrophilic shell that solubilizes the polymer in water, while the coreforms a hydrophobic microenvironment that encapsulates small hydrophobicmolecules. Unlike conventional micelles, however, the polymeric micellesof the present invention are thermodynamically stable because of thecovalent linkages between the polymer arms. The ability to encapsulatesmall molecules, the enhanced solubility and the lack of aggregationcharacterize the usefulness of these polymers as drug delivery systems.Candidate drugs, of which there are many, have aromatic orheteroaromatic moieties and carbonyl functionalities (e.g., amides andcarboxylates). The biocompatibility and biodegradability of thesepolymers further characterize their utility for drug delivery. Theexcellent water-solubility of these polymers makes intravenous injectionand oral administration of hydrophobic drug molecules possible. Forcontrolled release applications, the small size of these polymers, alongwith their enhanced thermodynamic stability, further characterizes theirutility.

Example 13 Interactions with Liposomes

Fusion of biological membranes is fundamental to a number ofphysiological and pharmacological processes. Because fusion is affectedby surfactant molecules, the colloidal polymers of the present inventionwere also expected to affect fusion processes. Liposomes, also referredto as vesicles, of dipalmitoyl-phosphatidylcholine (DPPC) are wellcharacterized in the literature and frequently used as models for cellmembranes (Attwood, D. and Florence, A. T. Surfactant Systems. TheirChemistry, Pharmacy and Biology, (London: Chapman Hall, 1983 293-387).

To monitor interactions of the polymeric micelle with DPPC liposomestructure, microdifferential scanning calorimetry (DSC) was utilized tomonitor thermotropic changes. After several days of stabilization, theDPPC vesicles showed two distinct transitions at 38° C. and 42° C.corresponding to small unilamellar vesicles (SUV) and large unilamellarvesicles (LUV), respectively. Addition of the polymeric micelle to thestabilized vesicles caused a rapid and complete shift in the DSC profilecorresponding to the complete conversion of SUVs to LUVs. Further, thepolymeric micelles promoted fusion processes to form morethermodynamically stable LUVs with a total enthalpy change of 12.8cal/g. Typically, the fusion event and conversion of SUVs to LUVs ispromoted by lowering the solution temperature well below the phasetransition to promote defects in the SUV structures. However, thesestudies demonstrate that addition of the polymeric micelles forceddefects in the SUV structure to enable fusion to occur at highertemperatures.

Confocal microscopy studies at 100× magnification revealed a cleardifference between solutions of DPPC liposomes with and without thepolymeric micelles. Differences could also be observed visually as thepolymer/liposome solution was clear while the liposome solution wasopaque. Further, after only one hour at room temperature, the liposomesaggregated and precipitated out of the aqueous media when the polymericmicelles were not present. By comparison, addition of the polymericmicelles to the liposome solution appeared to stabilize the liposomessuch that the solution remained translucent for at least 11 months whenstored at room temperature.

Using freeze fracture techniques, this stabilization was visualizedusing transmission electron microscopy. The average molecular size ofliposomes, polymeric micelles and polymeric micelle/liposome mixtures inaqueous media was determined as the ratio between the average diameterin microns. All samples were prepared with a concentration of 2 mg/ml.In liposome solutions, two populations existed with average sizes of0.10 μm and 0.50 μm. The average size of the polymeric micelles was 0.16μm. When the polymeric micelles were added to the liposome solutions,only one population was observed with an average size of 0.08 μm. Theseresults are consistent with other studies demonstrating that addition ofthe polymeric micelles stabilizes liposome structures.

Example 14 Polymer In Vitro Degradation

Hydrolytic degradation of polymer samples, in triplicate, was performedat 37° C. in a sodium phosphate buffer solution at pH 7.4. A smallamount of the buffer solution was taken at frequent time intervals up to12 months for analysis. Using both HPLC and GPC methods, no degradationof the polymer was observed under these storage conditions.

The enzyme papain was used to biodegrade polymeric micelles. Papain is aproteolytic enzyme with a defined structure and established properties.Function is optimal at pH 5.0 but also works in neutral and alkalinemedia. Papain is incompletely soluble in water, and practicallyinsoluble in most organic solvents, which simplifies the isolation andanalytical methods. Polymer degradation was monitored by following theappearance of degradation products such as mucic acid, hexanoic acid,MA(hex), tris(hydroxyphenyl)ethane and PEG by HPLC. To prepare samplesfor HPLC analysis, polymeric micelles were dissolved in buffered enzymesolution. At defined time intervals, the solution was extracted withethyl ether such that the polymer and the degradation products remainedin the organic phase while the enzyme remained in the aqueous solution.The organic phase was analyzed by HPLC using the refractive indexdetector and also evaluated by NMR and IR spectroscopies.

Results from these experiments indicate that the amide bond linking PEGto the polymer is cleaved first after two hours of incubation. The coremolecule was not observed up to 24 hours but was then rapidly released.Mucic acid and hexanoic acid were not detectable using UV or RIdetectors, but were observed using NMR and IR spectroscopic methods upto 24 hours.

Example 15 Evaluation of Cytotoxicity

In vitro toxicity characteristics of the polymeric micelles wereexamined in L-929 fibroblasts. The viability of the fibroblasts wasmonitored by counting live cells at 3, 7 and 21 days using polymerconcentrations of 1×10⁻⁴ M, 10⁻⁶ M, 10⁻⁷ M and 10⁻⁸ M. Cells which hadbeen incubated in solutions of polymeric micelles at the highestconcentration (10⁻⁴ M) did not survive at any time point. However,controls solutions which contained only PEG also had the same effect.All fibroblasts maintained in polymer solutions of lower concentrations(from 10⁻⁶ M to 10⁻⁸ M) survived and proliferated in the observed timeperiod.

The foregoing examples and description of the preferred embodimentshould be taken as illustrating, rather than as limiting, the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thespirit and scope of the invention, and all such modifications areintended to be included within the scope of the following claims.

1. A polymer having a structure selected from the group consisting of:R(—O—R₁), and R(—NH—R₁)_(x) wherein R(—O—)_(x) is a polyol moiety andR(—NH)_(x) is a polyamine moiety, with x being between 2 and 10,inclusive, and each R₁ independently has the structure:

wherein

is a divalent amino acid moiety with R₂ being a covalent bond or havingfrom 1 to 8 carbon atoms, and y and z are between 0 and 10, inclusive,provided that y and z are not both 0; wherein

is a divalent dicarboxylic acid moiety in which R₃ is an alkylene orcycloalkylene group containing from 1 to about 15 carbon atoms,substituted with a total of from 1 to about 10 hydroxyl groups, with atleast a portion of the hydroxyl groups being acylated with from 3 toabout 24 carbon atom carboxylic acids; and wherein R₄ is a poly(alkyleneoxide) having the structure:R₅—(R₆—O—)_(a)—R₆-Q- with R₅ being selected from the group consisting of1 to 40 carbon atom alkyl groups, —OH, —OR₇, —NH₂, —NHR₇, —NHR₇R₈,—C—OH, —C—OR₇, —C—O—C—R₇, —C—NH₂, —C—NHR₇ and —C—NR₇R₈; R₆, R₇ and R₈being independently selected from the group consisting of 2 to 40 carbonatom, straight-chain or branched alkylene groups; Q being a divalentlinkage moiety; and a being between 2 and 110, inclusive; with theproviso that when y is 0 and R is a 1,1,1-tris(hydroxyphenyl)ethanemoiety, the divalent dicarboxylic acid moiety is not an acylated mucicacid moiety.
 2. The polymer of claim 1, wherein x is 3 or
 4. 3. Thepolymer of claim 2, having the structure R(—NH—R₁)_(x), whereinR(—NH—)_(x) a polyamine moiety.
 4. The polymer of claim 2, having thestructure R(—O—R₁)_(x), wherein R(—O—)_(x) is a polyol moiety.
 5. Thepolymer of claim 4, wherein said polyol moiety is an aromatic polyolmoiety.
 6. The polymer of claim 5, wherein said polyol moiety is a1,1,1-tris(hydroxyphenyl)ethane moiety.
 7. The polymer of claim 1,wherein every hydroxyl group of said divalent dicarboxylic acid moietyis acylated with a 6 to 24 carbon atom carboxylic acid group.
 8. Thepolymer of claim 7, wherein y is
 0. 9. The polymer of claim 7, whereinsaid divalent dicarboxylic acid mucic acid moiety is a mucic acidmoiety.
 10. The polymer of wherein said polyol moiety is an aliphatic orcycloaliphatic polyol moiety.
 11. The polymer of claim 4, wherein saidpolyol moiety is a cyclic crown ether or cyclodextrin moiety.
 12. Thepolymer of claim 1, wherein said poly(alkylene oxide) is amethoxy-terminated poly(ethylene glycol) and Q is —NH—.
 13. The polymerof claim 1, wherein Q is —O—, or forms an anhydride linkage.
 14. Ahydrophobic molecule encapsulated in a polymer, said polymer having astructure selected from the group consisting of:R(—O—R₁)_(x) and R(—NH—R₁)_(x) wherein R(—O—)_(x) is a polyol moiety andR(—NH—)_(x) is a polyamine moiety, with x being between 2 and 10,inclusive, and each R₁ independently has the structure:

wherein

is a divalent amino acid moiety with R₂ being a covalent bond or havingfrom 1 to 8 carbon atoms, and y and z are between 0 and 10, inclusive,provided that y and z are not both 0; wherein

is a divalent dicarboxylic acid moiety in which R₃ is an alkylene orcycloalkylene group containing from 1 to about 15 carbon atoms,substituted with a total of from 1 to about hydroxyl groups, with atleast a portion of the hydroxyl groups being acylated with from 3 toabout 24 carbon atom carboxylic acids; and wherein R₄ is a poly(alkyleneoxide) having the structure:R₅—(—R₆—O—)_(a)—R₆-Q with R₆ being selected from the group consisting of1 to 40 carbon atom alkyl groups, —OH, —OR₇, —NH₂, —NHR₇, —NHR₇R₈,—C—OH, —C—OR₇, —C—O—C—R₇, —C—NH₂, —C—NHR₇, and —C—NR₇R₈; R₆, R₇ and R₈being independently selected from the group consisting of 2 to 40 carbonatom, straight-chain or branched alkylene groups; Q being a divalentlinkage moiety; and a being between 2 and 110, inclusive; with theproviso that when y is 0 and R is a 1,1,1-tris(hydroxyphenyl)ethanemoiety, the divalent dicarboxylic acid moiety is not a mucic acid moietyacylated with carboxylic acids having less than 6 carbon atoms.
 15. Theencapsulate of claim 14, wherein said hydrophobic molecule hasbiological or pharmaceutical activity.
 16. The encapsulate of claim 14,wherein said hydrophobic molecule is a fragrance, dye, protein orvitamin.
 17. The encapsulate of claim 14 having the structureR(—O—R₁)_(x), wherein R(—O—)_(x), is a polyol moiety.
 18. Theencapsulate of claim 17, wherein said polyol moiety is a1,1,1-tris(hydroxyphenyl)ethane moiety.
 19. The encapsulate of claim 17,wherein all of said hydroxyl groups of said dicarboxylic acid moiety areacylated with carboxylic acids having from 6 to 24 carbon atoms.
 20. Theencapsulate of claim 19, wherein y is
 0. 21. The encapsulate of claim19, wherein said divalent dicarboxylic acid moiety is a mucic acidmoiety.
 22. The encapsulate of claim 14, wherein said poly(alkyleneoxide) is a methoxy-terminated poly(ethylene glycol) and Q is —NH—. 23.A formulation comprising the encapsulate of claim 14 and an acceptablevehicle.
 24. The formulation of claim 23 wherein the accetable vehiclecomprises an aqueous solution, suspension, dispersion, emollient,lotion, cream, salve, balm or ointment.
 25. The formulation of claim 23,wherein the concentration of said encapsulate in said formulation isbetween about 10⁻¹⁰ M and 10⁶ M.
 26. The formulation of claim 25,wherein said encapsulate concentration is between about 10⁻⁸ M and about10⁶ M.
 27. The formulation of claim 23, wherein the hydrophobic moleculeof the encapsulate has biological or pharmaceutical activity.
 28. Theformulation of claim 23, wherein the hydrophobic molecule of theencapsulate is a dye, fragrance, protein, or vitamin.
 29. A method ofdelivering a hydrophobic molecule to a host comprising administering tothe host an encapsulate of claim
 14. 30. The method of claim 29 whereinthe host is an animal and the encapsulate is administered orally,subcutaneously, intramuscularly, intraperitoneaily or intravenously. 31.The method of claim 29 wherein the encapsulate is administered topicallyto the host.
 32. The method of claim 29 wherein the hydrophobic moleculeof the encapsulate is biologically or pharmaceutically active.
 33. Themethod of claim 29 wherein the hydrophobic molecule of the encapsulateis a dye, fragrance, protein or vitamin and the encapsulate isadministered topically.
 34. A transdermal device for the controlledrelease of a biologically or pharmaceutically active agent to the skinor mucosa of an animal, said device comprising a laminate of: (a) areservoir layer having a first surface and a second surface, and havingthe encapsulate of claim 15 incorporated therein. (b) a backing layerhaving an inner surface and an outer surface, wherein said secondsurface of said reservoir layer and said inner surface of said backinglayer are laminated together so that said encapsulate cannot permeatefrom said second surface of said reservoir layer through said outersurface of said backing layer; and (c) a pressure sensitive adhesivelayer coated on said first surface of said reservoir layer or said innersurface of said backing layer for affixing said laminate to said skin ormucosa of said host, so that said encapsulate is capable of beingcontinuously released from said first surface of said reservoir layerthereto.
 35. The device of claim 34, wherein said backing layercomprises one or more layers of an occlusive material.
 36. A method ofstabilizing a liposome or lipid-based formulation comprising adding tothe liposome or lipid-based formulation a polymer of claim 1.